The 0x7 Gene of Arabidopsis 1s Temporally and …soybeans, Kato et al. (1992) have identified three new lipox- ygenase isozymes. AI1 of these can be distinguished from the seed isozymes

Plant Physiol. (1994) 105: 385-393

The f 0x7 Gene of Arabidopsis 1s Temporally and Spatially Regulated in Germinating Seedlings'

Melissa A. Melan, Annette 1. D. Enriquez, and T. Kaye Peterman* Department of Biological Sciences, Wellesley College, Wellesley, Massachusetts 02 181

We examined the temporal and spatial expression patterns of the LOX7 gene during the development of Arabidopsis thaliana seedlings. Measurements of steady-state L O X 7 mRNA levels indi- cated that this gene is transiently expressed. during germination. LOX7 mRNA was not detected in seed that had imbibed (To) but reached a maximum leve1 by 1 d in both light- and dark-grown seedlings. l h e induction of the L O X 7 gene was not light dependent; however, mRNA levels were 4-fold greater in light-grown seedlings. lmmunoblot analysis of lipoxygenase protein levels and measurements of enzyme activity suggested that the induction of the LOX7 gene resulted in the production of functional lipoxygenase enzyme. Lipoxygenase protein was not present in dry seed or seed that had imbibed, but was first detected by immunoblot analysis after 1 and 2 d of growth in the light and dark, respectively. In both cases, lipoxygenase protein levels remained high for 2 d and then declined. Lipoxygenase activity paralleled the changes in protein levels. In situ hybridization studies revealed that the LOX7 gene is transiently expressed in the epidermis and the aleurone layer during germination. LOX7 mRNA levels were particularly high in the epidermis of the radicle and the adaxial side of the cotyledons. These resuits suggest that the L O X 7 gene product is produced specifically during early germination and plays a role in the functioning of the epidermis.

Lipoxygenases catalyze the hydroperoxidation of unsatu- rated fatty acids such as linoleic and linolenic acids, which contain a pentadiene double bond system (see Mack et al., 1986; Vick and Zimmerman, 1987; Hildebrand et al., 1988; Siedow, 1991, for reviews). The primary products of the lipoxygenase reaction are typically metabolized into molecules of known or suspected regulatory activity. In mammals, lipoxygenase-derived fatty acid hydroperoxides are precursors for the synthesis of a number of eicosanoids (e.g. pros- taglandins, prostacyclins, leukotrienes, lipoxins, and throm- boxans) that regulate specific cellular responses (Anderson, 1989). In higher plants, two major pathways have been described for the metabolism of fatty acid hydroperoxides. They are known collectively as the "lipoxygenase pathway" (Vick and Zimmerman, 1987). One branch of the lipoxygenase pathway produces traumatic acid, a compound that may be involved in wound responses in plant cells (Zimmerman and Coudron, 1979), as well as C6 volatiles such as trans-2- hexenal, which is antimicrobial and may play a role in pathogen defense (Croft et al., 1993). The second branch

This work was supported by National Science Foundation grants DCB-8904717 and IBN-9220254 to T.K.P.

* Corresponding author; fax 1-617-283-3642. 385

produces jasmonic acid, a molecule likely to serve a regulatory role in plant cells (Staswick, 1992; Sembdner and Parthier, 1993). At physiological concentrations jasmonates promote seed germination (Berestetzky et al., 1991; Ranjan and Lewak, 1992) and stimulate cell division (Ravnikar et al., 1992). In addition, it has also been suggested that jasmonates are involved in a wound- and pathogen-induced signal- transduction cascade (Farmer and Ryan, 1992).

The in vivo functions of lipoxygenase in higher plants are not clear. However, evidence has accumulated that indicates that plant lipoxygenases may play an important-role in a variety of processes, including stress responses (Bell and Mullet, 1991, 1993), pathogen defense (Croft et al., 1993; Melan et al., 1993), senescence, and the regulation of growth and development (Hildebrand et al., 1988; Siedow, 1991). Unfortunately, it has been difficult to determine the exact function of lipoxygenase in these processes, in part because the plants examined to date have large lipoxygenase gene families that encode a number of lipoxygenase isozymes (Siedow, 1991). In the hope of finding a more simple system for studying the physiological functions of lipoxygenase in higher plants, we initiated a molecular genetic and biochemical analysis of the lipoxygenases of Arabidopsis thaliana. A similar approach has been undertaken by Bell and Mullet (1993). A11 studies to date indicate that Arabidopsis contains a very simple lipoxygenase gene family. Only two members have been identified. We previously identified the LOXI gene of Arabidopsis (Melan et al., 1993) and Bell and Mullet (1993) have identified a second lipoxygenase gene in Arabidopsis, AtLox2, which is distinct in sequence and expression pattems from LOXI.

We are interested in the function of lipoxygenases during germination and ear!y development. Increases in lipoxygenase activity during germination have been reported for a number of plant species, including soybean (Holman, 1948; Hildebrand and Hymowitz, 1983; Park and Polacco, 1989; Kato et al., 1992), wheat (Guss et al., 1968), pea (Anstis and Friend, 1974), barley (Yabuuchi, 1976; Doderer et al., 1992), rice (Ohta et al., 1986), cucumber (Matsui et al., 1988, 1992), lupine (Beneytout et al., 1988), and Vicia sativa (Andrianari- son and Beneytout, 1992). For rice (Ohta et al., 1986), cucumber (Matsui et al., 1992), and soybean (Park and Polacco, 1989; Kato et al., 1992; Park et al., 1994), evidence consistent with the de novo synthesis of lipoxygenases during gennination has also been reported. In rice, the increase in lipoxygenase activity during germination is inhibited by cyclo- hexamide (Ohta et al., 1986), whereas in cucumber cotyledons the increase in activity has been correlated with an

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386 Melan e t al. Plant Physiol. Vol. 105, 1994

increase in lipoxygenase protein as determined by immuno- blotting (Matsui et al., 1992). In soybean, Park and Polacco (1989) have shown that at least two lipoxygenase isozymes, with isoelectric points distinct from the well-characterized seed lipoxygenase isozymes, appear in the hypocotyl/radicle axis during germination. Results from protein-labeling experiments suggested that these axis lipoxygenase species are newly synthesized during germination. Park et al. (1994) have also recently characterized two soybean lipoxygenase cDNA clones that are expressed in the axis during germination and are likely to encode the germination-associated lipoxygenase isozymes. In the cotyledons of germinating soybeans, Kato et al. (1992) have identified three new lipoxygenase isozymes. AI1 of these can be distinguished from the seed isozymes by ion-exchange chromatography, and the partia1 protein sequence of one (L-4) is distinct from the seed isozyme sequences.

In the studies presented here, we have examined the expression of the LOXZ gene of Arabidopsis during the germination and early development of light- and dark-grown seedlings. These results indicate that the LOXl gene is under strict temporal and spatial control during germination. The LOXZ gene is transiently expressed in the epidermis during the 1st d of development, the time during which the radicle emerges. We have also shown that the induction of the LOXl gene is correlated with the transient accumulation of functional lipoxygenase enzyme. These results suggest that the LOXZ gene product is important for a function of the epider- m i s at a precise time during development and provide the background required for “antisense” experiments, which will directly test this hypothesis.

MATERIALS AND METHODS

Plant Material

Arabidopsis thaliana ecotype Columbia seedlings were wet with 0.002% (v/v) Tween 20 and then surface sterilized by washing with 70% (v/v) ethanol for 1 min followed by two washes with sterile water and a 10-min treatment in 30% (v/v) bleach. The sterilized seeds were then extensively washed with sterile water, suspended in 1 mL of 0.1% (w/v) agarose/2500 seeds, and plated on defined mineral nutrient medium containing 0.6% (w/v) agarose (Estelle and Somer- ville, 1987) on 100 X 20 mm Petri plates. Approximately 2500 seeds were plated on each plate. The plates were covered and sealed with filter tape (Carolina Biological Sup- ply, Burlington, NC) to allow for gas exchange and then incubated in the light at 4OC for 72 h to break dormancy and synchronize germination. To samples were collected after the 4OC incubation. The plates were then transferred to a 24OC chamber and grown under continuous illumination (200-300 pE m-’ s-’) with a combination of fluorescent and incandes- cent lamps or under continuous darkness. Samples were collected at To and at various times thereafter and frozen in liquid Nz. Samples were ground to a powder in liquid NZ with a mortar and pestle and stored at -8OOC.

RNA lsolation and RNA Gel-Blot Analysis

Total RNA was isolated by phenol-SDS extraction and LiCl precipitation (Ausubel et al., 1987). RNA samples (10 pg)

were separated by electrophoresis on formaldeh yde-agarose gels (Ausubel et al., 1987), transferred to nylon filters (Bio- trans, ICN, Irvine, CA) by capillary transfer in 25 mM sodium phosphate, pH 6.5, and cross-linked to the filter by UV irradiation (200 pW cm-2 for 12-15 min). A parallel set of samples were run and stained with 1 pg/mL of ethidium bromide to verify even loading of samples. The filters were prehybridized for 1 to 2 h and then hybridized ovemight at 65OC with the full-length LOXl cDNA clone (Melan et al., 1993) labeled with [a-32P]dCTP by random prirning (Multi- prime IGt, Amersham, Arlington Heights, IL). This probe does nclt hybridize with the AtLox2 gene under these condi- tions. The filters were washed twice at room temperature in 0.5% (MI/V) BSA, 1 mM EDTA, 80 mM sodium phosphate (pH 7.2), and 5% (w/v) SDS followed by four 15-min washes at 65OC ir1 1 mM EDTA, 80 mM sodium phosphate (pH 7.2), and 1%~ (w/v) SDS (Church and Gilbert, 1984). The damp filters were exposed to Kodak X-AR5 film (Eastman Kodak, Rochester, NY) at -8OOC with a Cronex intensifying screen (Dupont).

Protein Extraction and lmmunoblot Analysis

Soluble protein extracts were prepared at 4OC by homog- enization of frozen tissue powders in 0.1 M sodiurn phosphate (pH 6.51) and 1% (w/v) polyvinylpolypyrrolidone. The ho- mogenaLtes were clarified by centrifuging twice ai 13,OOOg for 15 min each. An aliquot of the supematant was used for determination of the total protein concentration using the method of Bradford (1976) with BSA as a standard. For the preparation of gel samples, aliquots of the restilting super- natant were immediately mixed with an equal volume of 2 X SDS sample buffer (125 mM Tris-C1, pH 6.8, 4%’ [w/v] SDS, 20% [v/v] glycerol, 0.02% [w/v] bromphenol blue) and frozen in liqui’d NZ. The soluble proteins were resolved by SDS- PAGE (Laemmli, 1970) and then transferred ekrtrophoreti- cally from the gels to Immobilon-P polyvinylidene difluoride filters (IMillipore, Bedford, MA). The blots were blocked by incubation for 1 to 16 h in 10% (I&) powdered nonfat dry milk di:jsolved in TBS-T (20 mM Tris-C1, pH 7.6, 137 mM NaCl, 0.1% [v/v] Tween 20). The filters were washed three times for 10 min each in TBS-T and then incubated for 1 h with a Y:lO,OOO dilution of an Arabidopsis lipoxygenase antibody in TBS-T containing 5% (w/v) powdered nonfat dry milk. The antibody had been raised against a lipoxygenase- maltose binding fusion protein produced in Estherichia coli (Peterman et al., 1994). The filters were then washed three times for 10 min each in TBS-T with 5% (w/v) powdered nonfat tlry milk and incubated for 1 h with a 1 : l O O O dilution of a dcinkey anti-rabbit horseradish peroxidase secondary antibody. The filters were again washed three times for 10 min each in TBS-T with 5% (w/v) powdered nonfat dry milk. The resulting immune complexes were detected vrith the ECL immunodetection system (Amersham) according to the man- ufacturer’s instructions.

Lipoxyg,enase Activity Assays

Soluble protein extracts were prepared as described above except ,that the extraction buffer contained 0.1 M sodium



/.OX/ Gene Regulation during Arabidopsis Germination 387

phosphate (pH 6.5), 1% (w/v) polyvinylpolypyrrolidone, 1%(v/v) Tween 20, and 10 ITIM EDTA, and the BCA proteinassay (Pierce) was used to determine the total protein con-centration. The extracts were not frozen but rather usedimmediately for enzyme-activity assays. Lipoxygenase activ-ity was measured polarographically in a 2.0-mL reactionvessel fitted with a Clark-type O? electrode. The assays wereperformed at 25°C in 0.1 M sodium phosphate, pH 6.5, andinitiated by the addition of 1.3 mM linoleic acid emulsified inTween-20 according to the method of Surrey (1964).

Tissue Preparation and in Situ Hybridization

Seedlings were fixed for 24 h at room temperature with4% (w/v) paraformaldehyde in 50 mM Pipes, pH 7.2, con-taining 2 mM CaCl2, dehydrated through an ethanol series,cleared in xylenes, and embedded in paraffin (TissuePrep,Fisher Scientific). Sections (7 jum thick) were mounted ontoslides coated with 100 Mg/mL poly-L-Lys (Sigma). After de-paraffinization and rehydration, the tissue sections weretreated for 30 min with 1 /ig/mL proteinase K (Boehringer-Mannheim) in 100 mM Tris-Cl, pH 7.5, 50 HIM EDTA at 37°Cand then acetylated (Hayashi, et al., 1978) with 0.25% (v/v)acetic anhydride in 0.1 M triethanolamine for 5 min at roomtemperature. Following dehydration through ethanol and airdrying, the sections were hybridized overnight at 42°C withRNA probes prepared from a linearized Bluescript plasmidcontaining nucleotides 1258 to 2640 of the LOX1 cDNA(AAtLoxl-1; Melan et al., 1993). 35S-labeled sense and anti-sense riboprobes were synthesized with either T3 or T7 RNApolymerase (RNA Transcription Kit, Stratagene). Probes wereapplied to the sections at an average concentration of 4 X 107

cpm/mL in a hybridization solution containing 50% (v/v)formamide, 0.3 M NaCl, 10 mM Tris-Cl, pH 7.5, 1 mM EDTA,10% (w/v) dextran sulfate, lx Denhardt's solution (0.02%[w/v] Ficoll, 0.02% [w/v] PVP, 0.02% [w/v] BSA), and 100mM DTT. After hybridization, the slides were washed threetimes at room temperature in 4x SSPE (lx SSPE = 0.2 MNaCl, 10 mM NaH2PO4, 1 mM EDTA), and 5 mM DTT, thentreated for 30 min at 37°C with 25 jug/mL RNase in 500 mMNaCl, 10 mM Tris-Cl, pH 7.5, 1 HIM EDTA. The slides weresubsequently washed three times at room temperature with2x SSPE, 5 HIM DTT and then washed three times at highstringency with O.lx SSPE, 1 mM DTT at 57°C. Followingdehydration through an ethanol series containing 300 mMammonium acetate and air drying, the slides were coatedwith Kodak NTB2 autoradiography emulsion (Eastman Ko-dak) diluted 1:1 (v/v) with 600 mM ammonium acetate. Thehybridized tissues were exposed to the emulsion for 17 d at4°C and subsequently developed for 3 min in Kodak D-19developer (Eastman Kodak). Tissue sections were stainedwith 0.025% (w/v) toluidine blue O and coverslips weremounted with Permount (Fisher Scientific). Bright- and dark-field microscopic observations were made using an OlympusVanox microscope, and photographs were taken on KodakTMax 400 film developed with Kodak D-76 developer (East-man Kodak).

RESULTS

Induction of the LOX1 Gene during Germination

The LOX1 gene of Arabidopsis is induced upon bacterialpathogen attack and also by the phytohormones ABA andmethyl jasmonate (Melan et al., 1993). To determine if thisgene is also temporally regulated during early seedling de-velopment, LOX1 steady-state mRNA levels were determinedduring development of light- and dark-grown plants. Seedswere plated on a defined mineral nutrient medium (Estelleand Somerville, 1987) without Sue and incubated at 4°C for72 h to break dormancy. T0 samples were collected after the4°C incubation. The plants were then grown in either contin-uous light or dark at 24°C. Arabidopsis seedlings grown inthis manner are shown in Figure 1. Under these conditionsgermination frequencies were approximately 100% and de-velopment of the majority of seedlings was synchronous.

LOX1 steady-state mRNA levels were determined by RNAgel-blot hybridization analysis of total RNA isolated fromsamples collected at T0 and during the following 5 d (Fig. 2).Equivalent amounts of total RNA were loaded in each laneas determined by the ethidium bromide staining intensity ofthe rRNA bands. LOX1 mRNA was not detected at T0. LOX1steady-state mRNA levels increased dramatically during the1st d of development of both light- and dark-grown plants.In light-grown plants LOX1 mRNA was first detected at 12h, whereas in dark-grown plants it was not detected until 1d. In both light- and dark-grown seedlings LOX1 mRNAlevels were maximal at 1 d and then declined during subse-quent days of development. Maximal LOX1 mRNA levels inlight-grown plants were approximately 4-fold greater than

Figure 1. Early development of A. t/ia/iana ecotype Columbia seed-lings. Light- (L) and dark-grown (D) seedlings collected at T0, 1, 2,3, 4, and 5 d are shown from left to right. Scale bar = 3 mm.



388 Melan et al. Plant Physiol. Vol. 105, 1994

6h 12h 1d 2d 3d 4d 5d 60,

50-

Figure 2. iOXJ gene induction during Arabidopsis seedling devel-opment. Steady-state LOX1 mRNA levels were determined by RNAgel-blot hybridization analysis. Total RNA was extracted, separatedon formaldehyde gels, and blotted onto a nylon membrane. Equiv-alent amounts of RNA (10 Mg), as determined by ethidium bromidestaining of the ribosomal RNAs, were loaded for each time point.The membranes were probed with a LOX1 cDNA fragment 32Plabeled by the hexanucleotide random-priming procedure.

those observed in dark-grown plants. The appearance oflight- and dark-grown plants was identical for the 1st d (Fig.1). After 12 h the seed appeared swollen by comparison tothe To seed, and within 1 d the radicles had emerged.

Lipoxygenase Protein and Enzymic Activity LevelsIncrease during Germination

Lipoxygenase protein (Fig. 3) and enzyme activity (Fig. 4)levels were measured to determine if the observed inductionof the LOX1 gene results in the increased production offunctional lipoxygenase protein early in development and isthus physiologically relevant.

Lipoxygenase protein levels were measured by immunoblotanalysis of total protein extracts. Protein extracts were pre-pared from light- and dark-grown seedlings collected atintervals over a 6-d time course. Protein samples were sepa-rated by 10% SDS-PAGE and then transferred electro-phoretically from the gels to polyvinylidene difluoridemembranes. The blots were probed with an Arabidopsis li-poxygenase antibody that had been raised against a lipoxy-genase-maltose binding fusion protein produced in E. coli.The Arabidopsis lipoxygenase detected by this antibody is 98kD (Peterman et al., 1994). This is in agreement with the size

S To 6h 12h 1d 2d 3d 4d 5d 6d

Figure 3. Accumulation of lipoxygenase protein during early de-velopment of light- and dark-grown Arabidopsis seedlings. Lipoxy-genase protein levels, as a function of seedling age, were deter-mined by immunoblot analysis of total protein extracts (5 /ig/lane)from light- (L) and dark-grown (D) seedlings.

40

30'

20

0 1 2 3 4 5 6 7

Days

Figure 4. Lipoxygenase activity levels during early development oflight- and dark-grown Arabidopsis seedlings. Lipoxygenase activitylevels, as a function of seedling age, were measured using a Clark-type oxygen electrode in 0.1 M sodium phosphate, pH 6.5, withlinoleic acid as substrate. The data shown are the average of threeto four measurements.

of the LOX1 gene product predicted from the full-lengthcDNA sequence (Melan et al., 1993). In the resulting immu-noblots lipoxygenase protein was not detected in dry seed orat TO (Fig. 3). Lipoxygenase protein was first detected after 1and 2 d of growth in the light and dark, respectively. In bothlight- and dark-grown seedlings the lipoxygenase proteinlevels remained high for 2 d and then declined during sub-sequent days of development.

Lipoxygenase enzyme activity levels were also measuredduring the early development of light- and dark-grown Ara-bidopsis seedlings. The results are shown in Figure 4. Thelipoxygenase activity levels paralleled the levels of lipoxygen-ase protein detected by immunoblot analysis. In light-grownplants an approximately 13-fold increase in lipoxygenaseactivity, relative to that of T0 seed, was observed on the 1std of growth. Within 2 d a 15-fold increase in activity wasobserved. In dark-grown plants the increase in lipoxygenaseactivity was only 3-fold on the 1st d. Within 2 d of growthin the dark an 8-fold increase in activity was observed. Inboth light- and dark-grown plants, the maximal level ofactivity was observed after 2 d of growth. Lipoxygenaseactivity levels then declined during subsequent days ofdevelopment.

After 2 d of growth, the time of maximal lipoxygenaseenzyme activity, there were pronounced morphological dif-ferences between the light- and dark-grown seedlings (Fig.1). In the light-grown seedlings the cotyledons were greenand expanding and root growth and root-hair formation hadbegun. The dark-grown seedlings exhibited extensive hypo-cotyl elongation, with no cotyledon expansion or root-hairformation.

Spatial Expression of the LOX1 Gene during Germination

In situ hybridization analysis of sectioned plant materialwas used to elucidate the spatial expression patterns of the



LOXl Gene Regulation during Arabidopsis Germination 389

LOXl gene during germination. Light- and dark-grown plant material, collected at To and 1, 2, and 3 d, was fixed, embedded in paraffin, and cut into 7-pm sections. The sections were hybridized with either sense or antisense 35S-labeled LOXl RNA probes and examined by bright- and dark-field microscopy .

Longitudinal (Figs. 5, E and F, and 6C) and cross-sections (Fig. 5, G and H) from seed collected at To were hybridized with a LOXl antisense probe. No LOXl mRNA was detected in these sections, in agreement with the RNA gel-blot hybridization analysis (Fig. 2). Identical results were obtained with sections from seed collected at To hybridized with a LOXl sense probe (data not shown).

Hybridization of longitudinal (Figs. 5, A and B, and 6A) and cross-sections (Figs. 5, C and D, and 6B) from 1-d-old light-grown seedlings, with an antisense probe, revealed that the LOXl gene is highly expressed in epidermal cells within the 1st d of development. In dark-field micrographs of both longitudinal (Fig. 5A) and cross-section (Fig. 5C) hybridization, signal was evident throughout the embryos but was most concentrated in the epidermal cells of the radicle and the adaxial side of the cotyledons. Hybridization was also seen in the aleurone layer, which is closely associated with the innermost cell layers of the testa.

The location of the LOXl mRNA in specific cell layers is most evident in the high-magnification bright-field micrographs shown in Figure 6. The hybridization signal in the epidermal cells of the adaxial surface of the cotyledons (c) is shown in Figure 6A. Hybridization of the probe with the epidermal cells of the radicle (r) and the abaxial surface of a cotyledon (c) is seen in Figure 68. Hybridization signal over the aleurone layer of cells (a), a remnant of the endosperm (Vaughn and Whitehouse, 1971), is also shown in Figure 68. Similar results were obtained with sections from 1-d-old dark-grown seedlings, with the exception that the concentration of hybridization signal was not as great as in the light- grown seedlings (data not shown). LOXl mRNA was not detected in sections from 2- and 3-d-old seedlings hybridized with the antisense probe (data not shown).

No hybridization signal was detected in sections from 1-d light-grown plants hybridized with the LOXl sense probe (Fig. 5, I-L). These results provide strong evidence that the hybridization signal detected in sections hybridized with the antisense probe (Figs. 5, A-D, and 6, A and B) was due to specific hybridization with the LOXl mRNA.

DISCUSSION The LOXl gene was previously shown to be regulated by

pathogen attack and the phytohormones ABA and jasmonic acid (Melan et al., 1993). The experiments presented here have demonstrated that the LOXl gene is also under precise temporal and spatial control during seed germination. Taken together, these results suggest that the LOXZ gene product functions not only in plant stress responses but also in seed germination.

Expression of the L O X l Cene 1s under Precise Developmental Control in Germinating Arabidopsis Seedlings

We have measured LOXl steady-state mRNA levels during the development of both light- and dark-grown Arabidopsis

seedlings (Fig. 2). The LOXl gene was transiently expressed early in development. LOXl mRNA levels were maximal within the 1st d of development and then rapidly declined. These results indicate that expression of the LOXl gene is under very precise temporal control. Furthermore, these data suggest that the product of the LOXZ gene functions during a precise time in development. Similarly in soybean, two seedling axis lipoxygenase transcripts were not detected in the seed but accumulated during germination. Unlike the LOXl mRNA, however, these transcripts continue to accumulate during early seedling development (Park et al., 1994).

The lnduction of the LOXl Cene 1s followed by an lncrease in Functional Lipoxygenase Protein

The transient accumulation of the LOXl mRNA is correlated with an increase in enzymically active lipoxygenase protein. Measurements of lipoxygenase protein (Fig. 3) and enzyme activity (Fig. 4) levels revealed an increase in functional lipoxygenase protein in both light- and dark-grown Arabidopsis seedlings. It is likely that this increase in active lipoxygenase is due to the production of the LOXl-encoded protein. Of the two lipoxygenase genes identified in Arabi- dopsis, only the LOXl gene is highly expressed during germination. The AtLox2 gene is expressed in seedlings following gennination (Bell and Mullet, 1993) at levels at least 1 order of magnitude lower than the LOXI transcript (data not shown). These results suggest that the increase in LOXl mRNA results in the production of functional lipoxygenase enzyme and is thus physiologically significant.

Biochemical studies of the LOXl -encoded lipoxygenase will be essential for determining the role of this enzyme in germination. In mammalian cells lipoxygenase reaction products are precursors to a number of molecules that regulate specific cellular responses, and this is likely to be the case for plant cells as well (Siedow, 1991). Determination of the regiospe- cificity for hydroperoxidation of the germination-specific Arabidopsis lipoxygenase will provide information needed to determine the type of lipoxygenase metabolites that can possibly be produced. It will also be important to directly identify the lipoxygenase-derived metabolites that are produced in young seedlings. Such studies have provided a starting point for the characterization of the the germination- associated cotyledon lipoxygenases of soybean (Kato et al., 1992) and should provide valuable insights into the role of the LOXl -encoded lipoxygenase in gennination.

lnduction of the LOXl Cene 1s Not Light Dependent

LOXZ mRNA and lipoxygenase protein and activity levels were determined in both light- and dark-grown plants because in Sinapis alba, a close relative of Arabidopsis, lipoxygenase synthesis was reported to be down-regulated by light and controlled by phytochrome in the cotyledons of young seedlings (Oelze-Karow and Mohr, 1976). In addition, there is evidence that the phytochrome system controls the expression of soybean lipoxygenase genes (Maccarrone et al., 1991). The induction of the LOXl gene and production of functional Epoxygenase protein was not down-regulated in the light. Rather, light-grown seedling LOXZ mRNA levels were ap-



390 Melan et al. Plant Physiol. Vol. 105, 1994

Figure 5. Localization of LOX1 transcripts. A, Dark-field micrograph of a longitudinal section of a 1-d light-grown seedlingafter in situ hybridization with a LOX1 antisense probe. B, Bright-field micrograph of the same section shown in A. C,Dark-field micrograph of a cross-section of a 1-d light-grown seedling after in situ hybridization with a LOX1 antisenseprobe. D, Bright-field micrograph of the same section shown in C. E, Dark-field micrograph of a longitudinal section of aTo seed after in situ hybridization with a LOX1 antisense probe. F, Bright-field micrograph of the same section shown inE. C, Dark-field micrograph of a cross-section of a T0 seed after in situ hybridization with a LOX1 antisense probe. H,Bright-field micrograph of the same section shown in C. I, Dark-field micrograph of a longitudinal section of a 1-d light-grown seedling after in situ hybridization with a LOX1 sense probe. ), Bright-field micrograph of the same section shownin I. K, Dark-field micrograph of a cross-section of a 1-d light-grown seedling after in situ hybridization with a /.OX! senseprobe. L, Bright-field micrograph of the same section shown in K. Scale bar in L = 100 MM for all sections.



LOX1 Gene Regulation during Arabidopsis Germination 391

0

Figure 6. Localization of LOX1 transcripts to specific cell layers.Bright-field micrographs of sections after in situ hybridization witha LOX1 antisense probe. A, Longitudinal section of a 1-d light-grownseedling. Silver grains are most concentrated over the adaxial epi-dermis of the cotyledons (c). B, Cross-section of a 1-d light-grownseedling. The radicle (r), aleurone layer (a), and abaxial surface of acotyledon (c) are shown. Silver grains are most concentrated overthe radicle (r) epidermis and the aleurone cell layer (a). C, Longi-tudinal section of a T0 seed. No silver grains are seen in seedlingsat this stage of germination. Scale bar in C = 20 MM for all sections.

proximately 4-fold greater than those in dark-grown seed-lings. In addition, the LOX1 mRNA was detected 12 h earlierin the light-grown seedlings. Similarly, lipoxygenase proteinand activity levels increased earlier and to higher levels inlight-grown plants. It will be interesting to determine thereasons for these differences. It is possible that light enhancesexpression of the LOX1 gene directly. Alternatively, light mayexert an influence in a more indirect way by its photo-morphogenetic effects. Although l-d-old light- and dark-grown seedlings were indistinguishable, by 2 d the morpho-genetic effects of light were pronounced.

The LOX1 Gene Is Transiently Expressed in theEpidermis during Germination

The spatial distribution of the LOX1 mRNA was examinedby in situ hybridization. The results demonstrated that theLOX1 gene is expressed transiently in the epidermis and inthe aleurone layer during germination. No LOX1 mRNA wasdetected in seeds that had imbibed (To). However, within 1d the LOX1 mRNA was found at high levels in the epidermis,especially of the radicle and the adaxial side of the cotyledons.The LOX2 gene was also highly expressed in the aleuronelayer, which is intimately associated with the testa (Figs. 5and 6). A low level of expression was detected throughoutthe embryo. In sections of 2- and 3-d-old seedlings no LOX1mRNA was detected. This is the first localization of a specificplant lipoxygenase mRNA.

Several immunolocalization studies of soybean lipoxygen-ase proteins have been reported (Vernooy-Gerritsen et al.,1983; Tranbarger et al., 1991; Grimes et al., 1992). However,multiple lipoxygenase isozymes are present in soybeans andit is likely that the antibodies used for all of these studiescross-reacted with a number of these species. Vernooy-Gerritsen et al. (1983) immunolocalized lipoxygenase proteinsin germinating soybean seeds. They found a high signalthroughout the cotyledons during the 1st d of germinationdue to the seed lipoxygenases. Within 3 d lipoxygenaseprotein was detected in the abaxial hypodermis, the vascularbundle sheaths, and the epidermis. Tranbarger et al. (1991)showed that in soybean leaves lipoxygenase proteins werelocalized primarily in the vacuoles of paraveinal mesophyllcells. A much lower level of lipoxygenase was also detectedin the epidermal cells. In methyl jasmonate-treated soybeanseedlings lipoxygenase proteins accumulated in the shoottips, primary leaves, epicotyls, hypocotyls, and cotyledons(Grimes et al., 1992). Especially high levels of lipoxygenaseprotein were seen in the epidermal cells of the cotyledonsand the cortical cells of the hypocotyl.

The expression of the LOX1 gene in the aleurone layer isintriguing. This cell layer, which is derived from the endo-sperm during embryogenesis, is typical of seeds of the Bras-sicaceae (Vaughn and Whitehouse, 1971). Its function duringseed germination, however, is unknown.

A number of genes that are expressed in the epidermis atsome point in the life cycle of the plant, including membersof the phenylammonia lyase and chalcone synthase genefamilies (Schmelzer et al., 1989), lipid transfer protein genes(Sossountzov et al., 1991; Sterk et al., 1991), genes involvedin flower pigmentation (Jackson et al., 1991; Goodrich et al.,1992), and soybean Pro-rich cell-wall proteins (Wyatt et al.,1992), or genes that are epidermis specific (Clark et al., 1992)have been identified. It will be interesting to determine if theregulatory sequences of the LOX1 gene share homology withother germination-specific and/or epidermally expressedgenes. From such an analysis it may be possible to identifyputative regulatory sequences required for gene expression,specifically in the epidermis and/or during germination.

The epidermis controls water loss and gas exchange andprovides mechanical and chemical defense against pathogenand predator attack. In addition, it has been postulated thatplant organ growth is controlled by the epidermis (Nick et



392 Melan e t al. Plant Physiol. Vol. 105, 1994

al., 1990). The epidermal location of the LOXl mRNA and its transient accumulation in young seedlings suggest that lipoxygenase contributes to one of these functions of the epidermis during germination. One possibility is that the LOXl gene product is required for the production of molecules that function prophylactically in the protection of young seedlings from pathogens. It is interesting to note that the mRNAs of severa1 other defense-related genes, which encode enzymes of the phenylpropanoid pathway, are localized to the epidermis in uninfected plants (Schmelzer e t al., 1989) and accumulate transiently during germination (Kubasek et al., 1992). It has been postulated that the phenylpropanoid pathway is required for the production of flavanoids, which protect the young seedlings from UV light or pathogens (Kubasek et al., 1992). Another possibility is that the LOXl gene product generates fatty acid hydroperoxide precursors to molecules that regulate the growth or expansion of the epidermis. Interestingly, jasmonic acid, a lipoxygenase- derived metabolite, stimulates germination in some species (Berestetzky et al., 1991; Ranjan and Lewak, 1992).

ACKNOWLEDCMENTS

We are especially grateful to Dr. Kathleen Dunn and Jaqueline Heard for their assistance with the in situ hybridizations. We also thank summer students Dorothy Huang, Holly Pieck, and Heather Ryback for technical assistance, and Drs. Erin Bell and Joseph Polacco for communicating results prior to publication.

Received September 20, 1993; accepted Februaj 8, 1994. Copyright Clearance Center: 0032-0889/94/105/0385/09.

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